The present invention generally relates to an assay for identifying inhibitors of the papilloma virus (PV), particularly human papilloma virus (HPV). In particular, the present invention provides a novel probe in a competitive assay to identify HPV inhibitors. More particularly, the present invention relates to the synthesis and use of a probe that binds with specificity to the transactivation domain (TAD) of HPV E2 to form a complex therewith, and which is capable of being displaced by inhibitors of HPV.
Papillomaviruses are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been identified in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, horses, and dogs. The first papillomavirus was described in 1933 as cottontail rabbit papillomavirus (CRPV). Since then, the cottontail rabbit as well as bovine papillomavirus type 1 (BPV-1) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human there are more than 75 types of papillomavirus that have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases such as cervical carcinomas.
There are more than 25 HPV types that are implicated in anogenital diseases, these are grouped into xe2x80x9clow riskxe2x80x9d and xe2x80x9chigh riskxe2x80x9d types. The low risk types include HPV type 6 and type 11 and induce mostly benign lesions such as condyloma acuminata (genital warts) and low grade squamous intraepithelial lesions (SIL). In the United States, 1% of the sexually active population has genital warts of which 90% is attributed to HPV-6 and HPV-11.
The high risk types are associated with high grade SIL and cervical cancer and include most frequently HPV types 16, 18, 31, 33, 35, 45, 52, and 58. The progression from low-grade SIL to high-grade SIL is much more frequent for lesions that contain high risk HPV-16 and 18 as compared to those that contain low risk HPV types. In addition, only four HPV types are detected frequently in cervical cancer (types 16, 18, 31 and 45). About 500,000 new cases of invasive cancer of the cervix are diagnosed annually worldwide.
The life cycle of PV is closely coupled to keratinocyte differentiation. Infection is believed to occur at a site of tissue disruption in the basal epithelium. Unlike normal cells, the cellular DNA replication machinery is maintained as the cell undergoes vertical differentiation. As the infected cells undergo progressive differentiation the viral genome copy number and viral gene expression in turn increase, with the eventual late gene expression and virion assembly in terminally differentiated keratinocytes and the release of viral particles.
The coding strands for each of the papillomavirus contain approximately ten designated translational open reading frames (ORFs) that have been classified as either early ORFs or late ORFs based on their location in the genome. E1 to E8 are expressed early in the viral replication cycle, and two late genes (L1 and L2) encode the major and minor capsid proteins respectively. The E1 and E2 gene products function in viral DNA replication, whereas E5, E6 and E7 are expressed in connection with host cell proliferation. The L1 and L2 gene products are involved in virion structure. The function of the E3 and E8 gene products is uncertain at present.
Studies of HPV have shown that proteins E1 and E2 are the only two viral proteins that are necessary for viral DNA replication in vitro and in vivo, in addition to the host DNA replication machinery. This requirement is similar to that of bovine papillomavirus type 1 (BPV-1). Indeed, there is a high degree of similarity between E1 and E2 proteins and the ori-sequences of all papillomaviruses (PV) regardless of the viral species and type. Evidence emanating from studies of BPV-1 have shown that E1 possesses ATPase and helicase activities that are required in viral DNA replication.
The E2 protein is a transcriptional activator that binds to E1 protein and forms a complex that binds specifically to the ori sequence (Mohr et al., 1990, Science 250:1694-1699). It is believed that E2 enhances binding of E1 to the BPV origin of replication (Seo et al., 1993, Proc. Natl. Acad. Sci., 90:2865-2869). In HPV, Lui et al. suggested that E2 stabilizes E1 binding to the ori (1995, J. Biol. Chem., 270(45):27283-27291). The HPV-16 transactivation domain (TAD) of E2 has been described in J. E. Burns et al., 1998 (Acta Cryst. D54, 1471-1474) and amino acids 1-190 were found to be required and sufficient for E1 binding (Yasugi et al., 1997, J. Virol. 71, 891-899).
To thwart this disease, a chemical entity that would interfere with or inhibit viral DNA replication is therefore desirable. Previously described methods to evaluate inhibitors of the E1-E2 interaction (U.S. Pat. No. 5,925,516 and Titolo et al. 1999, J. Virol. 73, 5282-5293) have relied on the production of full-length E1 and E2 proteins. HPV E2 and especially E1 have been difficult to obtain in sufficient quantity and purity for effective drug screening (White et al., 2001, J. Biol. Chem., 276(25), 22426-22438; Rocque et al., 2000, Protein, Expression Purif. 18, 148-159). Furthermore, one common assay for this interaction involves measuring the cooperative binding of E1 and E2 to double-stranded DNA referred to herein as the E2-dependent E1 DNA binding assay (Titolo et al. 1999, J. Virol. 73, 5282-5293). This method is highly sensitive to salt concentration and pH, as is well known to be true in general for protein-DNA interactions. Furthermore, protein-DNA interactions are sensitive to inhibition by nonspecific DNA intercalators (Lai et al., 1992, Proc Natl. Acad. Sci. USA, 89(15):6958-62).
One family of chemical entities that inhibit HPV replication is disclosed in WO 02/50082 published Jun. 27, 2002. The mechanism of action of these inhibitors was elucidated and they were found to inhibit the E1-E2 interaction by binding to the E2 TAD. We have therefore rationalized that, used as probes, these could be displaced by test compounds that also inhibit or disrupt the E1:E2 interaction, an interaction that is critical for the complex to bind to DNA and proceed with viral replication. Validation of this rationale could be obtained by testing the inhibitors identified in the present assay with a well known E2-dependent E1-DNA binding assay.
The present invention therefore provides a probe and a novel displacement assay for screening for potential inhibitors of papilloma viral replication. Advantageously, this displacement assay of the present invention is easy to use and inexpensive and amenable to adjustments in salt concentration or pH levels. This type of assay is also amenable to a high sensitivity and a high throughput format, and uses a protein that has a low molecular weight, which is easy to purify.
It is a further advantage of the present invention to provide a probe that binds to the transactivation domain of HPV E2 with a high affinity, and which is displaced by inhibitors of HPV.
The present description refers to a number of documents, the content of which is herein incorporated by reference.
In a first embodiment, the invention provides a probe of formula (I) or its enantiomers or diastereoisomers thereof: 
wherein:
A is a 5- or 6-membered homocyclic ring, or a 5- or 6-membered heterocyclic ring containing 1 or more heteroatoms selected from N, O and S;
X is H and W is OH; or X and W together form a carbonyl group or an epoxide;
R1 is H; or one or two substituents independently selected from the group consisting of: hydroxy, halo, lower alkyl, lower alkoxy, lower thioalkyl, haloalkyl (e.g. trifluoromethyl), or xe2x80x94C(O)R2 wherein R2 is lower alkyl, aryloxy or benzyloxy;
Y is phenyl optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 is lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy, nitrile or trifluoromethyl, and R6 is lower alkyl, lower cycloalkyl, lower alkoxy, hydroxy or trifluoromethyl; said phenyl ring being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
or Y is a heterocycle (Het) containing one or more heteroatom selected from N, O or S, said Het optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and R6 are as defined above; said Het being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
or Y is ethylene-phenyl, said ethylene moiety being optionally mono-substituted with lower alkyl, wherein said phenyl ring is optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and R6 are as defined above; said phenyl ring being optionally fused with a saturated or unsaturated 4- to 6-membered ring optionally containing a heteroatom selected from N, O and S;
or Y is ethylene-Het, said ethylene moiety being optionally mono-substituted with lower alkyl, wherein Het is optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and Re are as defined above; said Het being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
R3 is selected from the group consisting of: lower alkyl, lower cycloalkyl, lower alkylene, aryl or lower aralkyl, all of which optionally mono- or di-substituted with:
lower alkyl, lower cycloalkyl, haloalkyl, halo, CN, azido, lower alkoxy, (lower alkyl)acyl, C1-6 thioalkyl, C1-6 alkylsulfonyl, NHC(O)-lower alkyl, NHC(O)-aryl, NHC(O)xe2x80x94O-lower alkyl, NHC(O)O-aryl, aryl, aryloxy, hydroxy, nitro, amino, or Het, said Het optionally mono- or di-substituted with lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy, nitrile, trifluoromethyl, C(O)R6 wherein R6 is as defined above;
said lower cycloalkyl, aryl, lower aralkyl or Het being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S; and
R4 is a carboxylic acid, a salt or an ester thereof;
or a derivative thereof;
wherein said derivative is a probe of Formula (I) labeled with a detectable label or an affinity tag, wherein wavy lines represent bonds of unspecified stereochemistry; and wherein said probe binds to the transactivation domain of HPV E2 and is capable of being displaced by a potential inhibitor thereof: or a derivative thereof, wherein said derivative is a probe of formula (I) labeled with a detectable label or an affinity tag, wherein wavy lines represent bonds of unspecified stereochemistry; and wherein said signal is selected from: fluorescence, resonance energy transfer, time resolved fluorescence, radioactivity, fluorescence polarization, change in the intrinsic spectral properties, luminescence and plasma-resonance; whereby a modulation in said signal is an indication that said test compound binds to said transactivation domain.
Alternatively, the first embodiment of the invention provides compounds having the following formulae, selected from the group consisting of: 
wherein R1, A, X, W, Y, R3 and R4 are as defined above.
Compounds of formula I may also be represented by forms (2) and (3): 
wherein R1, A, X, W, Y and R3 are as defined above.
As will be recognized by persons skilled in the art, the compounds in forms (2) and (3) are readily converted to compounds of formula (I) in form (1). Without wishing to be bound by theory, it is believed that the compounds of formula (I) are in equilibrium between forms (1), (2) or (3) depending on the solvent and the pH in which they are dissolved. It has however been demonstrated that compounds of formula (I) are biologically active in form (1), and that the compounds in forms (2) and (3) will hydrolyze in conditions reproducing mammalian plasma (pH 7.4) to yield biologically active form (1).
According to a second embodiment of the invention, there is provided an assay for the identification of inhibitors of HPV replication, comprising:
a) contacting a transactivation domain of HPV E2 protein with a probe of formula I as defined above to form an E2:probe complex and measuring a signal from said probe to establish a base line level;
b) incubating a E2:probe complex with a test compound and measuring the signal from said probe in said complex; and
c) comparing the signal from step a) with the signal from step b);
whereby a modulation in said signal is an indication that said test compound binds to said transactivation domain.
As will be understood by a person skilled in the art, steps a) and b) in the above mentioned assay may be carried out in sequence or in parallel i.e. the control signal from the E2:probe can be measured prior to the addition of the test compound or the control signal can be measured in a well distinct from the well where the E2:probe complex is mixed with the test compound.
An alternative aspect of this second embodiment provides an assay for the identification of inhibitors of HPV replication, comprising:
a) contacting a transactivation domain of HPV E2 protein with a probe of formula I as defined above to form an E2:probe complex and measuring a signal from said probe to establish a base line level;
b) incubating a E2 protein with a test compound;
bxe2x80x2) adding a probe of formula (I) to said mixture of E2 and test compound from step b) and measuring the signal from said probe; and
c) comparing the signal from step a) with the signal from step bxe2x80x2);
whereby a modulation in said signal is an indication that said test compound binds to said transactivation domain.
As will be understood by a person skilled in the art, steps a) and b) in the above mentioned assays are usually carried out in parallel i.e. the control signal from the E2:probe is measured in a well distinct from the well where the E2:test compound is mixed with the probe.
As will be understood by a person skilled in the art, the probe of formula (I) used for the present assay can be replaced without undue burden by any alternative compound found in WO 02/50082 incorporated herein by reference.
As will be understood by a person skilled in the art, modulation in the signal means either a decrease or an increase in the signal. Usually, modulation in the signal will be observed as a decrease in signal.
According to a third embodiment of the invention, there is provided the use of a probe according to formula (I) in the development of an assay for identifying inhibitors of HPV replication.
According to a fourth embodiment of the invention, there is provided a kit for testing compounds that potentially bind to the transactivation domain of HPV, said kit comprising a probe according to formula (I); and instructions on how to use said probe for identifying test compounds binding to said transactivation domain.
According to a fifth embodiment of the invention, there is provided a reagent for testing compounds that potentially bind to the transactivation domain of HPV E2, said reagent comprising a E2:probe complex as defined above.